Manipulation of self-incompatibility in plants

ABSTRACT

The present invention relates to methods for controlling hybridization in plants and producing hybrid plants. The present invention also relates to nucleic acids encoding amino acid sequences for self-incompatibility (SI) proteins in plants, and the use thereof for the manipulation of SI, including seed production, in plants, particularly of the Poaceae family. The present invention also relates to kits, compositions, constructs and vectors including such nucleic acids, and related polypeptides, regulatory elements and methods. The present invention also relates to expression of self-gamete recognition genes in plants and to related nucleic acids, constructs, molecular markers and methods.

FIELD OF THE INVENTION

The present invention relates to methods for controlling hybridization in plants and methods for producing hybrid plants. The present invention also relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for self-incompatibility proteins in plants, in particular through self-gamete recognition in plants of grass and cereal species, and the use thereof for the manipulation of SI, including seed production, in plants. The present invention also relates to kits, compositions, constructs and vectors including such nucleic acids, and related polypeptides, regulatory elements and methods.

The present invention also relates to expression of self-gamete recognition genes in plants and to related nucleic acids, constructs, molecular markers derived from nucleic acids and related methods.

BACKGROUND OF THE INVENTION

The phenomenon through which some flowering plant species are unable to successfully reproduce through self-pollination has been termed ‘self-incompatibility’ (SI). A definition of SI was accepted as proposed by Lundqvist, being ‘the inability of a fertile hermaphrodite seed-plant to produce zygotes after self-pollination’. This phraseology was in order to distinguish between SI and the effect of post-fertilisation barriers. SI has been described in about 30% of all flowering plants, and is the most important system for prevention of self-fertilization. The key part of this system is self-recognition, in which the pistil discriminates between self- or non-self-pollen to either inhibit or permit pollen tube germination and/or elongation and resulting fertilisation.

There are multiple genetic mechanisms that regulate and enable the system, SI does not represent a unique system. The molecular basis of the plant SI mechanism has been well studied in several groups of dicotyledonous plant species. Self-incompatibility locus (S locus) genes were identified in winged tobacco (Nicotiana elate) and Brassica rapa L. (syn. campestris) in the late 1980s.

Subsequent biological investigations using multiple approaches have identified SI factors, to help elucidate the molecular basis of SI mechanisms in these species.

Plant species within the Poaceae (grass and cereal) family, can display an obligate outbreeding reproductive habit controlled by a two-locus (termed S and Z) gametophytic SI system, in which the pollen genotype is autonomously controlled by its own genetic constitution. This system is conserved between allogamous Poaceae species, such as wild barley (Hordeum bulbosum L.) and cereal rye (Secale cereale L.) and has been found to be widely conserved within the family, but is expected to be genetically and mechanistically distinct from the well-characterised single-locus SI mechanisms of dicotyledonous plants.

The Poaceae-specific mechanism prevents self-fertilisation through arrest of self-generated gamete pollen tube elongation at the stigmatic surface. Although several of the key molecular signals involved in dicot-specific SI systems have been identified, the molecular basis of the Poaceae system remains unknown.

Free calcium concentrations are essential for directed cell growth in pollen tubes in many species. The role calcium plays was initially identified as increasing cell wall rigidity and regulating permeability. However, the specific concentration of calcium in the cell is critical, as free calcium is typically kept at c. 100 nm due to cellular metabolism being based on free phosphates, and if free cytosolic calcium levels elevate over this concentration, interference with the energy status of the cell will result due to the formation of calcium salts. Studies of calcium gradients within the pollen tube have identified an increased gradient at the active growing tip, and the increase has been postulated to be absorbed by the cellular growth.

Previously, calcium has been identified to play a role in regulation of the SI system in the Poaceae family. Treatment with calcium channel blockers (lanthanum and verapamil) has been demonstrated to inhibit the perennial ryegrass SI mechanism.

By treating excised stigmas with the chemical blocking agents self pollen was able to germinate.

The S and Z loci of perennial ryegrass (Lolium perenne L.) have been assigned to linkage groups (LGs) 1 and 2 respectively, in regions of known macrosynteny with the genomes of the inbreeding cereal species rice (Oryza sativa L.) and wheat (Triticum aestivum L.). Fine-structure mapping of the Poaceae SI loci was performed for blue canary grass (Phalaris coerulescens L.) and cereal rye (Secale cereale L.), and the candidate gene-containing regions were delimited to 0.26 cM and 1.5 cM intervals for the S and Z loci, respectively. The presence of gene-associated (cDNA-based) markers in these studies permitted comparative analysis to define map colinearity around the SI loci for related self-incompatible and self-compatible Poaceae species. The proposed 1.5 cM Z-containing region exhibited microsynteny with a BAC clone (OSJNBa0070011: GenBank Acc. No. AL606445) from rice chromosome 4 c. 125 kb in length, to which 12 predicted genes have been assigned.

For outbreeding crops, understanding and regulation of SI mechanisms can simplify and accelerate breeding procedures. For example, knowledge of the Solanaceae S-RNase-based SI system informed a method for almond cultivar development through use of a previously-characterised self-compatible (S_(c)) mutant line. The program involved introduction of the S_(c) allele into existing almond varieties for enhanced fixation of genes for favourable oil content and fatty acid composition. Inbreeding also enables simpler maintenance of agronomically elite lines.

In the standard semi-hybrid breeding scheme for outbreeding pasture crop species, two cultivar groups are intercrossed to generate a progeny population with increased yield owing to heterosis. However, around half the progeny are derived from an intracross within each parental group and do not receive the benefit of heterosis. Heterosis in forage mass has been also reported in perennial ryegrass and other grass crop species using the standard semi-hybrid breeding system.

In a new scheme with SI genotyping technology, an SI-allele restricted population is established, and intercrossing between the restricted population and a cultivar group generates a progeny population with a higher ratio of hybrid progeny. An experiment with red clover proved higher hybrid ratios in progenies and improved seed yields when the restricted populations were used.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction or that this prior art could reasonably be expected to be ascertained, understood and regarded as relevant by a person skilled in the art.

It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.

SUMMARY OF THE INVENTION

Applicants have used an extensive and inclusive approach involving both genomics and transgenic modification for molecular dissection of the SI pathway in monocots. Spatio-temporal profiles of gene expression, comparative genomics, BAC clone sequencing and whole-exome sequencing suggests that the components of the SI pathway (the S and Z genes) may be encoded by a collection of genes from within the Poaceae family that have not been previously characterised as having these functions in ryegrass.

Applicants have found that modification or selection of the genes located at the S and Z loci of outbreeding plants of the Poaceae family is an attractive strategy for controlling pollination and fertilization by repression or activation of the SI mechanism. Applicants have also found that modification or selection of the genes located at the S and Z loci of plants of the Poaceae family may be used to control hybridization or to produce hybrid plants in higher numbers than conventional breeding approaches. By identifying the nucleic acid sequences of the genes, transgenic modification through down-regulation or through inducible expression enables hybrid breeding schemes to be enabled. Modification of the nucleic acids through targeted gene disruption, by the use of transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs), mediating cleavage of specific target sites in the nucleic acid, leading to micro-deletions and insertions within the endogenous nucleic acid sequence, also enables control of fertilisation. The use of molecular markers derived from inherent variation originating from within the nucleic acid sequences may also provide a predictive means for control of fertilisation.

A perennial ryegrass BAC-based genomic library composed of 50,304 (131×384-well plates) BAC clones (average insert size=113 kb) has been constructed to support contig assembly, and estimated to correspond to c. 3.4 genome equivalents (Spangenberg et al. 2005; Forster et al. 2008). A combination of fine-structure genetic linkage mapping and physical genome characterisation enables implementation of map-based cloning to isolate perennial ryegrass SI genes.

In addition, whole-genome sequencing has been performed to generate genic contigs from the perennial ryegrass genome that can assist with gene identification and nucleic acid characterisation. These efforts have been undertaken on a single plant genotype that has been clonally propagated to provide sufficient source material (Cogan et al. 2012a, Forster et al. 2012). A global gene expression (transcriptome) profile has also been extensively generated and characterised through sequencing of RNA nucleic acids from the same plant as was used for the genome sequence (Cogan et al. 2012b, Forster et al. 2012).

Identification and characterisation of the S and Z genes may enable establishment of novel breeding methodology for perennial ryegrass.

For example, plants of the Poaceae family may be transformed with a gametophytic Z gene nucleic acid wherein (1) transformation with said Z gene specific nucleic acid transforms a self incompatible plant of the Poaceae family into a self compatible plant or (2) transformation with said Z gene specific nucleic acid transforms a self compatible plant of the Poaceae family into a self incompatible plant. In a preferred embodiment, the gametophytic Z gene may encode a 26S proteasome subunit, a zinc finger protease, a no-pollen (NOP) polypeptide, or an ubiquitin-specific protease, such as an ubiquitin-specific protease 22.

For example, plants of the Poaceae family may be transformed with a gametophytic S gene nucleic acid wherein (1) transformation with said S gene specific nucleic acid transforms a self incompatible plant of the Poaceae family into a self compatible plant or (2) transformation with said S gene specific nucleic acid transforms a self compatible plant of the Poaceae family into a self incompatible plant. In a preferred embodiment, the gametophytic S gene may encode a Cullin, a glutamate receptor or precursor thereof, or a seven-in-absentia homologue (SIAH).

Accordingly, in a first aspect, the present invention provides a composition or kit for hybridization or self-incompatibility (SI) control in plants, said composition or kit including:

-   -   a first nucleic acid or nucleic acid fragment encoding a SI         polypeptide, wherein said first nucleic acid or nucleic acid         fragment is isolated from or corresponds to a gene from the Z         locus of a plant of the Poaceae family; and     -   a second nucleic acid or nucleic acid fragment encoding a SI         polypeptide, wherein said second nucleic acid or nucleic acid         fragment is isolated from or corresponds to a gene from the S         locus of a plant of the Poaceae family.

Preferably said first and second nucleic acids are substantially purified or isolated.

In a preferred embodiment, the first nucleic acid or nucleic acid fragment may be a gametophytic Z gene.

In a preferred embodiment, the second nucleic acid or nucleic acid fragment may be a gametophytic S gene.

In a particularly preferred embodiment the first and second nucleic acids or nucleic acid fragments may be selected from the group of nucleic acids and nucleic acid fragments as hereinafter described.

For example, the Z gene may encode a 26S proteasome subunit, a zinc finger protease, a no-pollen (NOP) polypeptide, or an ubiquitin-specific protease, such as an ubiquitin-specific protease 22, as hereinafter described.

For example, the S gene may encode a Cullin, a glutamate receptor or precursor thereof, or a seven-in-absentia homologue (SIAH), as hereinafter described.

In a further preferred embodiment, the first and second nucleic acids or nucleic acid fragments may be included in a construct or vector, as hereinafter described.

In a further aspect of the present invention there is provided a method for controlling hybridization in a plant or for producing hybrid plants, said method including:

-   -   establishing or identifying a first plant strain with a first Z         locus haplotype and a first S locus haplotype;     -   establishing or identifying a second plant strain with a second         Z locus haplotype and a second S locus haplotype; and     -   crossing said plant strains to produce hybrid plants;     -   wherein said haplotypes are selected so that the first plant         strain is heterozygous at both the S and Z loci and said second         plant strain is homozygous at one of the S and Z loci and         heterozygous at the other of the S and Z loci.

Preferably the first and second plant strains are plants of the Poaceae family. More preferably they are grass species, particularly pasture grasses such as ryegrass (Lolium) or fescue (Festuca), more particularly perennial ryegrass (Lolium perenne L.) or tall fescue (Festuca arundinaceum, otherwise known as Lolium arundinaceum).

Preferably the haplotypes are from genes according to the present invention, as hereinafter described.

In a further aspect of the present invention there is provided a method of manipulating self-incompatibility in a plant, said method including introducing into said plant an effective amount of a nucleic acid, construct and/or vector according to the present invention.

In a preferred embodiment the method involves altering the SI status of the plant.

In a preferred embodiment, the method may include introducing into said plant:

-   -   a first nucleic acid or nucleic acid fragment encoding a SI         polypeptide, wherein said first nucleic acid or nucleic acid         fragment is isolated from or corresponds to a gene from the Z         locus of a plant of the Poaceae family; and     -   a second nucleic acid or nucleic acid fragment encoding a SI         polypeptide, wherein said second nucleic acid or nucleic acid         fragment is isolated from or corresponds to a gene from the S         locus of a plant of the Poaceae family.

For example, the Z gene may encode a 26S proteasome subunit, a zinc finger protease, a no-pollen (NOP) polypeptide, or an ubiquitin-specific protease, such as an ubiquitin-specific protease 22, as hereinafter described.

For example, the S gene may encode a Cullin, a glutamate receptor or precursor thereof, or a seven-in-absentia homologue (SIAH), as hereinafter described.

The present invention also contemplates co-expressing a nucleic acid of the present invention with a gene encoding a mediator or modulator of SI activity.

By SI status is meant the ability or inability of a fertile hermaphrodite seed-plant to produce zygotes after self-pollination.

By a ‘mediator or modulator of SI activity’ is meant a molecule that enhances or otherwise modifies expression, activity or function of SI in a plant cell, plant callus, plant, seed or other plant part. For example, the mediator or modulator of SI activity may improve pollen tube growth, or enhance action or activity of the SI mechanisms.

By “an effective amount” it is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.

Using the methods and materials of the present invention, self-incompatibility may be induced, increased, decreased, repressed or otherwise altered, in a transformed plant relative to an untransformed control plant, for example by incorporating additional copies of a sense nucleic acid of the present invention, preferably to overexpress the polypeptide or in sense suppression. They may be decreased or otherwise altered, for example by incorporating an antisense nucleic acid of the present invention.

In a further aspect, the present invention provides a method for altering the SI status of a plant, said method including identifying a gene encoding a polypeptide which is active in the SI pathway of the plant and up-regulating or down-regulating expression of said gene to repress or induce the SI mechanism in said plant. Preferably said gene is a nucleic acid according to the present invention. Preferably the plant is as hereinbefore described.

By ‘up-regulating’ expression of said gene is meant increasing expression of said gene and, as a result, the protein encoded by the gene, in a plant relative to a control plant.

By ‘down-regulating’ expression of said gene is meant decreasing expression of said gene and, as a result, the protein encoded by the gene, in a plant relative to a control plant.

The up-regulation or down-regulation may be carried out by methods known to those skilled in the art. For example, a gene may be up-regulated by incorporating additional copies of a sense copy of the gene. A gene may be down-regulated, for example, by incorporating an antisense nucleic acid, a frame-shifted or otherwise modified sense copy of the gene, or nucleic acid encoding interfering RNA (RNAi). Up or down regulation may also be achieved through the use of transcription activator-like effector nucleases or zinc-finger nucleases, mediating cleavage of specific target sites in the nucleic acid, leading to micro-deletions and insertions within the endogenous nucleic acid sequence.

Techniques for incorporating the genetic constructs of the present invention into plant cells are known to those skilled in the art. Such techniques include high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. Cells incorporating the genetic constructs of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well know in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced either sexually or asexually, using methods well known in the art.

By ‘repressing the SI mechanism’ of a plant is meant reducing the tendency of the plant to inhibit pollen tube elongation and resulting fertilisation of self-pollen.

By ‘activating the SI mechanism’ of a plant is meant introducing the tendency of the plant to inhibit pollen tube elongation and resulting fertilisation of self-pollen.

In a further aspect, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a plant self-incompatibility (SI) protein, complements thereof, sequences antisense thereto, and functionally active fragments and variants thereof. Preferably, the nucleic acid or nucleic acid fragment encodes a polypeptide selected from the group consisting of a proteasome subunit, more particularly a 26S proteasome subunit, a Cullin (Cullins are molecular scaffolds responsible for assembling ubiquitin E3 ligases, more particularly RING-based E3 ubiquitin ligases), a glutamate receptor or precursor thereof, a zinc finger protease, a no-pollen (NOP) polypeptide, a seven-in-absentia homologue (SIAH), and a ubiquitin-specific protease, more particularly a ubiquitin-specific protease 22.

The nucleic acid or nucleic acid fragment may be isolated from or correspond to a gene from a plant of the Poaceae family. In a preferred embodiment the nucleic acid or nucleic acid fragment may be isolated from or correspond to a gene from a grass species, particularly a pasture grass such as ryegrass (Lolium) or fescue (Festuca), more particularly perennial ryegrass (Lolium perenne L.) or tall fescue (Festuca arundinaceum, otherwise known as Lolium arundinaceum).

By ‘nucleic acid’ is meant a chain of nucleotides capable of genetic information. The term generally refers to genes or functionally active fragments or variants thereof and or other sequences in the genome of the organism that influence its phenotype. The term ‘nucleic acid’ includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA or microRNA) that is single- or double stranded, optionally containing synthetic, non-natural or altered nucleotide bases, synthetic nucleic acids and combinations thereof.

Nucleic acids according to the invention may be full-length genes or part thereof, and are also referred to as “nucleic acid fragments” and “nucleotide sequences” in this specification. For convenience, the expression “nucleic acid or nucleic acid fragment” is used to cover all of these.

By ‘substantially purified’ is meant that the nucleic acid is free of the genes, which, in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived, flank the nucleic acid. The term therefore includes, for example, a nucleic acid which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote;

or which exists as a separate molecule (e.g. a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a nucleic acid which is part of a hybrid gene encoding additional polypeptide sequence. Preferably, the substantially purified nucleic acid is 90%, more preferably 95%, even more preferably 98% pure.

The term “isolated” means that the material is removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.

Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term “consensus contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acids or nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a plant self-incompatibility (SI) protein, or complementary or antisense to a sequence encoding a plant SI protein, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NOS: 1 to 70;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NOS: 71 to 140;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

Preferably, the SI protein is selected from the group consisting of a proteasome subunit, more preferably a 26S proteasome subunit, a Cullin, a glutamate receptor or precursor thereof, a zinc finger protease, a protein containing C2 and GRAM amino acid domains, more preferably a protein involved in signal transduction, membrane trafficking, and/or membrane-coupled processes, more preferably a protein encoded by a NOP gene, a SIAH, and a ubiquitin-specific protease, more preferably a ubiquitin-specific protease 22.

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a proteasome subunit, more preferably a 26S proteasome subunit, or complementary or antisense to a sequence encoding a proteasome subunit, more preferably a 26S proteasome subunit, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 58 and FIG. 3;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 128;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a Cullin, or complementary or antisense to a sequence encoding a Cullin, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 20 and FIG. 5;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 90;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a glutamate receptor or precursor thereof, or complementary or antisense to a sequence encoding a glutamate receptor or precursor thereof, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 39 and FIG. 7;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 109;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a zinc finger protease, or complementary or antisense to a sequence encoding a zinc finger protease, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 62 and FIG. 9;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 132;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a C2 and GRAM domain containing polypeptide, preferably a no pollen (NOP) polypeptide, or complementary or antisense to a sequence encoding a C2 and GRAM domain containing polypeptide, preferably a no pollen (NOP) polypeptide, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 59 and FIG. 11;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 129;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a seven-in-absentia homologue, or complementary or antisense to a sequence encoding a seven-in-absentia homologue, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 40 and FIG. 13;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 110;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

In a preferred embodiment, the present invention provides a substantially purified or isolated nucleic acid or nucleic acid fragment encoding a ubiquitin-specific protease, more preferably a ubiquitin-specific protease 22, or complementary or antisense to a sequence encoding a ubiquitin-specific protease, more preferably a ubiquitin-specific protease 22, said nucleic acid or nucleic acid fragment including a nucleotide sequence selected from the group consisting of:

-   -   (a) the sequences shown in SEQ ID NO: 54 and FIG. 15;     -   (b) a nucleotide sequence encoding the polypeptide shown SEQ ID         NO: 124;     -   (c) complements of the sequences recited in (a) and (b);     -   (d) sequences antisense to the sequences recited in (a) and (b);     -   (e) functionally active fragments of the sequences recited in         (a), (b), (c) and (d); and     -   (f) functionally active variants of the sequences recited in         (a), (b), (c), (d) and (e).

The present invention encompasses functionally active fragments and variants of the nucleic acids of the present invention. By ‘functionally active’ in relation to the nucleic acid is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of manipulating SI in a plant. For example, it may be capable of manipulating a proteasome in a plant, more particularly a proteasome subunit, even more particularly a 26S proteasome subunit. For example, it may be capable of manipulating an E3 ubiquitin ligase in a plant, more particularly a Cullin. For example, it may be capable of manipulating influx channels in a plant, more particularly glutamate receptors. For example, it may be capable of manipulating ubiquitin-specific protease activity in a plant, more particularly zinc finger protease activity. For example, it may be capable of manipulating signal transduction, membrane trafficking, and/or membrane-coupled processes in a plant. For example, it may be capable of manipulating SIAH in a plant. For example it may be capable of manipulating a ubiquitin-specific protease in a plant, more particularly a ubiquitin-specific protease 22.

Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative nucleic acid changes.

Particularly preferred fragments include fragments of the nucleic acid sequences which include hypervariable regions of the gametophytic gene in sense or anti sense orientation, and functionally active variants of these fragments, see FIGS. 17 to 37.

Preferably the fragment has a size of at least 20 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 200 nucleotides, more preferably at least 500 nucleotides.

In a particularly preferred embodiment, the fragment or variant may include a sequence shown in FIGS. 17 to 37 hereto.

By ‘conservative nucleic acid changes’ is meant nucleic acid substitutions that result in conservation of the amino acid in the encoded protein, due to the degeneracy of the genetic code. Such functionally active variants and fragments also include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by another one of the same class, the classes being as follows:

-   -   Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp     -   Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln     -   Acidic: Asp, Glu     -   Basic: Lys, Arg, His

Other conservative amino acid substitutions may also be made as follows:

-   -   Aromatic: Phe, Tyr, His     -   Proton Donor: Asn, Gln, Lys, Arg, His, Trp     -   Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln

In a further aspect of the present invention, there is provided a genetic construct including one or more nucleic acids according to the present invention.

In a preferred embodiment the genetic construct may include a chimeric sequence comprising a nucleic acid according to the present invention and a gene encoding a mediator or modulator of SI activity.

In another preferred embodiment, the genetic construct may include:

-   -   a first nucleic acid or nucleic acid fragment encoding a SI         polypeptide, wherein said first nucleic acid or nucleic acid         fragment is isolated from or corresponds to a gene from the Z         locus of a plant of the Poaceae family; and     -   a second nucleic acid or nucleic acid fragment encoding a SI         polypeptide, wherein said second nucleic acid or nucleic acid         fragment is isolated from or corresponds to a gene from the S         locus of a plant of the Poaceae family.

The term “genetic construct” as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. Preferably the genetic construct is a recombinant nucleic acid molecule. In general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

By a ‘chimeric sequence’ is meant a hybrid produced by recombinant means through expression of a fusion gene including two or more linked nucleic acids which originally encoded separate proteins, or functionally active fragments or variants thereof.

By a ‘fusion gene’ is meant that two or more nucleic acids are linked in such a way as to permit expression of the fusion protein, preferably as a translational fusion. This typically involves removal of the stop codon from a nucleic acid sequence coding for a first protein, then appending the nucleic acid sequence of a second protein in frame. The fusion gene is then expressed by a cell as a single protein. The protein may be engineered to include the full sequence of both original proteins, or a functionally active fragment or variant of either or both.

In a preferred embodiment, the genetic construct according to the present invention may be a vector.

By a ‘vector’ is meant a genetic construct used to transfer genetic material to a target cell. The term vector encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.

The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens; derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable or integrative or viable in the target cell.

In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.

By a ‘promoter’ is meant a nucleic acid sequence sufficient to direct transcription of an operatively linked nucleic acid sequence.

By ‘operatively linked’ is meant that the nucleic acid(s) and a regulatory sequence, such as a promoter, are linked in such a way as to permit expression of said nucleic acid under appropriate conditions, for example when appropriate molecules such as transcriptional activator proteins are bound to the regulatory sequence. Preferably an operatively linked promoter is upstream of the associated nucleic acid.

By ‘upstream’ is meant in the 3′→5′ direction along the nucleic acid.

The promoter and terminator may be of any suitable type and may be endogenous to the target cell or may be exogenous, provided that they are functional in the target cell.

The promoter used in the constructs and methods of the present invention may be a constitutive, tissue specific or inducible promoter. For example, the promoter may be a constitutive cauliflower mosaic virus (CaMV35S) promoter for expression in many plant tissues, an inducible ‘photosynthetic promoter’ (e.g. ribulose 1,5-bisphosphate), capable of mediating expression of a gene in photosynthetic tissue in plants under light conditions, or a tissue specific promoter such as a seed specific promoter, for example from a gene selected from the group consisting of Brassica napus napin gene, Zea mays zein 4 gene, Orysa sativa PR602 gene and Triticum aestivum glutelin gene.

A variety of terminators which may be employed in the genetic constructs of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the (CaMV)35S polyA and other terminators from the nopaline synthase (nos) and the octopine synthase (ocs) genes.

The genetic construct, in addition to the promoter, the gene and the terminator, may include further elements necessary for expression of the nucleic acid, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (nptII) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. The genetic construct may also contain a ribosome binding site for translation initiation. The genetic construct may also include appropriate sequences for amplifying expression.

Those skilled in the art will appreciate that the various components of the genetic construct are operably linked, so as to result in expression of said nucleic acid. Techniques for operably linking the components of the genetic construct of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.

In a still further aspect, the present invention provides a substantially purified or isolated regulatory element capable of causing expression of an exogenous gene in plant cells. Preferably the regulatory element is isolated from a nucleic acid or nucleic acid fragment encoding a plant self-incompatibility (SI) protein and functionally active fragments and variants thereof. Preferably, the regulatory element is isolated from a nucleic acid or nucleic acid fragment encoding a proteasome subunit, more particularly a 26S proteasome subunit, a Cullin, a glutamate receptor or precursor thereof, a zinc finger protease, a polypeptide with both C2 and GRAM amino acid domains, more preferably a no-pollen (NOP) gene, a SIAH, or a ubiquitin-specific protease, more preferably a ubiquitin-specific protease 22.

The regulatory element may be a nucleic acid molecule, including DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.

Preferably the regulatory element includes a promoter. In a preferred embodiment, the regulatory element includes a proteasome subunit gene promoter, more preferably a 26S proteasome subunit gene promoter. In another preferred embodiment the regulatory element includes a Cullin gene promoter. In another preferred embodiment the regulatory element includes a glutamate receptor or precursor gene promoter. In another preferred embodiment the regulatory element includes a zinc finger protease gene promoter. In another preferred embodiment the regulatory element includes a promoter from a polypeptide with both C2 and GRAM amino acid domains, more preferably a promoter from a no-pollen (NOP) gene. In another preferred embodiment the regulatory element includes a SIAH gene promoter. In another preferred embodiment the regulatory element includes a ubiquitin-specific protease gene promoter, more preferably a ubiquitin-specific protease 22 gene promoter.

Preferably the regulatory element may be isolated from or correspond to a regulatory element from a plant of the Poaceae family. In a preferred embodiment the regulatory element may be isolated from or correspond to a regulatory element from a grass species, particularly a pasture grass such as ryegrass (Lolium) or fescue (Festuca), more particularly perennial ryegrass (Lolium perenne L.) or tall fescue (Festuca arundinaceum, otherwise known as Lolium arundinaceum).

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from a 26S proteasome subunit gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 3; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 2335 of FIG. 3.

In another particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from a Cullin gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 5; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 294 of FIG. 5.

In another particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from glutamate receptor or precursor gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 7; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 788 of FIG. 7.

In another particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from zinc finger protease gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 9; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 625 of FIG. 9.

In another particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from a NOP gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 11; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 7924 of FIG. 11.

In another particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from a SIAH gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 13; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 124 of FIG. 13.

In another particularly preferred embodiment of this aspect of the invention, the regulatory element includes a promoter from a ubiquitin-specific protease gene from perennial ryegrass.

Preferably the regulatory element includes a promoter element of the sequence shown in FIG. 15; or a functionally active fragment or variant thereof, including hypervariable regions. The person skilled in the art will understand that the promoter element is located upstream of the ATG start codon shown at position 6784 of FIG. 15.

By “functionally active” in this context is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of causing expression of a transgene in plant cells, particularly of the reproductive tissues. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the regulatory element. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, even preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Preferably the fragment has a size of at least 100 nucleotides, more preferably at least 150 nucleotides, most preferably at least 200 nucleotides.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of:

Nucleotides 0 to 2334 of FIG. 3,

Nucleotides 500 to 2334 of FIG. 3, and

Nucleotides 1000 to 2334 of FIG. 3;

or a functionally active fragment or variant thereof.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of: Nucleotides 0 to 293 of FIG. 5, and functionally active fragments and variants thereof.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of: Nucleotides 0 to 787 of FIG. 7, and functionally active fragments and variants thereof.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of: Nucleotides 0 to 624 of FIG. 9, and functionally active fragments and variants thereof.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of:

Nucleotides 0 to 7923 of FIG. 11,

Nucleotides 6968 to 7923 of FIG. 11, and

Nucleotides 7468 to 7923 of FIG. 11,

or a functionally active fragment or variant thereof.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of: Nucleotides 0 to 123 of FIG. 13, and functionally active fragments and variants thereof.

In a particularly preferred embodiment of this aspect of the invention, the regulatory element includes a nucleotide sequence selected from the group consisting of:

Nucleotides 0 to 6783 of FIG. 15,

Nucleotides 5087 to 6783 of FIG. 15,

Nucleotides 5587 to 6783 of FIG. 15, and

Nucleotides 6087 to 6783 of FIG. 15;

or a functionally active fragment or variant thereof.

By an “exogenous gene” is meant a gene not natively linked to said regulatory element. In certain embodiments of the present invention the exogenous gene is also not natively found in the relevant plant or plant cell.

The exogenous gene may be of any suitable type. The exogenous gene may be a nucleic acid such as DNA (e.g. cDNA or genomic DNA) or RNA (e.g. mRNA), and combinations thereof. The exogenous gene may be a gene capable of manipulating SI in a plant, or be a fragment or variant (such as an analogue, derivative or mutant) thereof which is capable of manipulating SI in a plant. Such variants include nucleic acid sequences which are antisense to said target gene or an analogue, derivative, mutant or fragment thereof. The transgene may code for a protein or RNA sequence depending on the target condition and whether down or up-regulation of gene expression is required.

The regulatory element according to the present invention may be used to express exogenous genes to which it is operatively linked in the production of transgenic plants. Preferably the regulatory element is used for gene expression in reproductive tissues of the plant.

Preferably, the genetic constructs of the present invention are substantially purified or isolated, as hereinbefore described. By ‘substantially purified’, in the current context, is meant that the genetic construct is free of the genes, which, in the naturally-occurring genome of the organism from which the nucleic acid or promoter of the invention is derived, flank the nucleic acid or promoter. The term therefore includes, for example, a genetic construct which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g. a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a genetic construct which is part of a hybrid gene encoding additional polypeptide sequence. Preferably, the substantially purified genetic construct is at least approximately 90% pure, more preferably at least approximately 95% pure, even more preferably at least approximately 98% pure.

As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the genetic construct in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical assays (e.g. GUS assays), thin layer chromatography (TLC), northern and western blot hybridisation analyses.

The genetic constructs and vectors of the present invention may be incorporated into a variety of plants, preferably monocotyledons, preferably of the Poaceae family, such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, sugarcane, wheat and barley.

The genetic constructs of the present invention may be introduced into plants by any suitable technique. Techniques for incorporating the genetic constructs of the present invention into plant cells (for example by transduction, transfection, transformation or gene targeting) are well known to those skilled in the art. Such techniques include Agrobacterium-mediated introduction, Rhizobium-mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos, biolistic transformation, Whiskers transformation, and combinations thereof. The choice of technique will depend largely on the type of plant or fungus to be transformed, and may be readily determined by an appropriately skilled person. For transformation of protoplasts, PEG-mediated transformation is particularly preferred.

Cells incorporating the genetic constructs of the present invention may be selected, as described below, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.

In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a vector or construct, nucleic acid or nucleic acid fragment of the present invention. Preferably the plant cell is a transformed plant cell.

By a ‘transformed plant cell’ is meant a plant cell which has undergone transformation.

By ‘transformation’ is meant the transfer of nucleic acid into a plant cell.

By a ‘transgene’ is meant a nucleic acid suitable for transforming a plant cell.

The plant cell, plant, plant seed or other plant part may be from any suitable species. In a preferred embodiment the plant cell, plant, plant seed or other plant part may be from a monocotyledon, preferably of the Poaceae family, such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, sugarcane, wheat and barley.

The present invention also provides a plant, plant seed or other plant part, or a plant extract derived from a plant cell or plant of the present invention and preferably including e.g. transformed with, a vector or construct, nucleic acid or nucleic acid fragment, or regulatory element of the present invention.

The nucleic acids or nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous SI proteins from the same or other plant species, using sequence-dependent protocols, such as methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction, ligase chain reaction).

For example, other 26S proteasome subunit genes, Cullin genes, glutamate receptor or precursor genes, zinc finger protease genes, NOP genes, SIAH genes, or ubiquitin-specific protease genes may be isolated directly by using all or a portion of the nucleic acids or nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. Moreover, the entire sequences may be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in protocols to amplify longer nucleic acids or nucleic acid fragments encoding homologous genes from DNA or RNA. For example, polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid sequences of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad USA 85:8998, the entire disclosure of which is incorporated herein by reference) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Using commercially available 3′ RACE and 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments may be isolated (Ohara et al. (1989) Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217, the entire disclosures of which are incorporated herein by reference). Products generated by the 3′ and 5′ RACE procedures may be combined to generate full-length cDNAs.

In a further aspect of the present invention there is provided a substantially purified or isolated SI polypeptide. Preferably, the SI polypeptide is selected from the group consisting of a proteasome subunit, more particularly a 26S proteasome subunit, a Cullin, a glutamate receptor or precursor thereof, a zinc finger protease, a polypeptide including both C2 and GRAM amino acid domains, more preferably a polypeptide encoded by a no-pollen (NOP) gene, a SIAH, and a ubiquitin-specific protease, more preferably a ubiquitin-specific protease 22.

The SI polypeptide may be isolated from or correspond to a polypeptide from a plant of the Poaceae family. In a preferred embodiment the SI polypeptide may be isolated from or correspond to a polypeptide from a grass species, particularly a pasture grass such as ryegrass (Lolium) or fescue (Festuca), more particularly perennial ryegrass (Lolium perenne L.) or tall fescue (Festuca arundinaceum, otherwise known as Lolium arundinaceum).

In a preferred embodiment, the present invention provides a substantially purified or isolated SI polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequences shown in SEQ ID NOS: 71 to 140 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NOS: 1         to 70 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated proteasome subunit polypeptide, more particularly a 26S proteasome subunit polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 128 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 58         and FIG. 3 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated Cullin polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 90 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 20         and FIG. 5 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated glutamate receptor polypeptide or precursor thereof, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 109 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 39         and FIG. 7 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated zinc finger protease polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 132 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 62         and FIG. 9 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated NOP polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 129 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 59         and FIG. 11 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated SIAH polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 110 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 40         and FIG. 13 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

In a preferred embodiment, the present invention provides a substantially purified or isolated ubiquitin-specific protease polypeptide, said polypeptide including an amino acid sequence selected from the group consisting of:

-   -   (a) sequence shown in SEQ ID NO: 124 hereto;     -   (b) polypeptides encoded by the sequences shown in SEQ ID NO: 54         and FIG. 15 hereto;     -   (c) functionally active fragments of the sequences recited         in (a) and (b); and     -   (d) functionally active variants of the sequences recited in         (a), (b) and (c).

The present invention encompasses functionally active fragments and variants of the polypeptides of the present invention. By ‘functionally active’ in this context is meant that the fragment or variant has one or more of the biological properties of the corresponding protein from which the fragment or variant is derived. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned sequence to which the fragment or variant corresponds, more preferably at least approximately 90% identity, more preferably at least approximately 95% identity, most preferably at least approximately 98% identity. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence.

By ‘conservative amino acid substitutions’ is meant the substitution of an amino acid by another one of the same class, the classes being as follows:

-   -   Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp     -   Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln     -   Acidic: Asp, Glu     -   Basic: Lys, Arg, His

Other conservative amino acid substitutions may also be made as follows:

-   -   Aromatic: Phe, Tyr, His     -   Proton Donor: Asn, Gln, Lys, Arg, His, Trp     -   Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln

Preferably the fragment has a size of at least 10 amino acids, more preferably at least 20 amino acids, more preferably at least 50 amino acids, more preferably at least 100 amino acids, more preferably at least 200 amino acids.

In a particularly preferred embodiment, the fragment or variant may include a sequence shown in FIGS. 38 to 58 hereto.

In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are known to those skilled in the art.

Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides may be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins including the amino acid sequences. These antibodies may be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest.

In a still further aspect of the present invention there is provided a method of isolating a nucleic acid or nucleic acid fragment of the present invention said method including sequencing nucleic acid fragments from a nucleic acid library.

The nucleic acid library may be of any suitable type and is preferably a cDNA library.

The nucleic acid or nucleic acid fragment may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction.

The sequencing may be performed by techniques known to those skilled in the art.

In a still further aspect, the present invention involves identifying variation in the sequence of a gene encoding a polypeptide which is active in the SI pathway of a plant and deploying such variants as molecular markers. More particularly, the method includes determining the specific genetic constitution of a plant within the Poaceae family at the S and Z loci through analysis of genetic variation at the S and Z loci using methods known to those skilled in the art. This genetic variation may be in regions surrounding the SI genes and may be used in a proxy manner. Examples of sequence variation within the genes and their encoded polypeptides are shown in FIGS. 17 to 58.

Accordingly, the present invention provides use of a nucleic acid or nucleic acid fragment of the present invention or a SNP thereof as a molecular genetic marker.

More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in grasses such as Lolium perenne. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention may be used as molecular genetic markers in plant improvement in relation to SI control or manipulation. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in grasses such as Lolium perenne.

As used herein, except where the context requires otherwise, the term “comprise” and variations of the term, such as “comprising”, “comprises” and “comprised”, are not intended to exclude further additives, components, integers or steps.

As used herein, except where the context requires otherwise, the singular forms “a”, “an” and “the” include plural aspects.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present invention will now be more fully described with reference to the accompanying examples and figures. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention description above.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

In the figures:

FIG. 1. Comparative genetic ideogram of the S region delimited in Lolium perenne L. in comparison to the model genomes of Oryza sativa and Brachypodium distachion. Genes identified in common between Oryza sativa and Brachypodium distachion are indicated by joining lines. Assembled fragments of sequenced BAC clones from Lolium perenne L. are indicated along with their predicted location within the comparative genome map and their gene content. Gene content of the Lolium perenne L. nucleotide sequences are documented as orthologous genes based on the Oryza numerical numbering, with a Lp prefix.

FIG. 2. Comparative genetic ideogram of the Z region delimited in Lolium perenne L. in comparison to the model genomes of Oryza sativa and Brachypodium distachion. Genes identified in common between Oryza sativa and Brachypodium distachion are indicated by joining lines. Assembled fragments of sequenced BAC clones from Lolium perenne L. are indicated along with their predicted location within the comparative genome map and their gene content. Gene content of the Lolium perenne L. nucleotide sequences are documented as orthologous genes based on the Oryza numerical numbering, with a Lp prefix.

FIG. 3. SEQ ID NO: 162, Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpOs06g0607800 26S proteasome subunit gene (SEQ ID NO: 58) The initial codon (ATG) of the zinc finger protease gene is shown in bold italic underline.

FIG. 4. Map of transformation vector containing the Lolium perenne ZmUbi_LpOs06g0607800_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIG. 5. SEQ ID NO: 163, Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpOs05g0149600 Cullin gene. (SEQ ID NO: 20) The initial codon (ATG) of the Cullin gene is shown in bold italic underline.

FIG. 6. Map of transformation vector containing the Lolium perenne ZmUbi_LpOs05g0149600_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIG. 7. SEQ ID NO: 164. Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpOs06g0680500 Glutamate Receptor (LpGlu1) gene. (SEQ ID NO: 39) The initial codon (ATG) of the glutamate receptor gene is shown in bold italic underline.

FIG. 8. Map of transformation vector containing the Lolium perenne ZmUbi_LpGlu1_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIG. 9. SEQ ID NO: 165, Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpOs04g0648500 Ubiquitin-specific protease 22 including a zinc finger protease gene. (SEQ ID NO: 62) The initial codon (ATG) of the zinc finger protease gene is shown in bold italic underline.

FIG. 10. Map of transformation vector containing the Lolium perenne ZmUbi_LpOs04g0648500_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIG. 11. SEQ ID NO: 166, Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpOs06g0607900 No-Pollen (LpNOP) gene. (SEQ ID NO: 59) The initial codon (ATG) of the LpNOP gene is shown in bold italic underline.

FIG. 12. Map of transformation vector containing the Lolium perenne ZmUbi_LpNOP_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIG. 13. SEQ ID NO: 167. Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpOs05g0152900 Seven-In-Absentia Homolog (LpSIAH) gene. (SEQ ID NO: 40). The initial codon (ATG) of the LpSIAH gene is shown in bold italic underline.

FIG. 14. Map of transformation vector containing the Lolium perenne ZmUbi_LpSIAH_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIG. 15. SEQ ID NO: 168, Nucleic acid sequence of the genomic clone that contains the Lolium perenne LpTC116908 gene. (SEQ ID NO: 54) The initial codon (ATG) of the LpTC116908 gene is shown in bold italic underline.

FIG. 16. Map of transformation vector containing the Lolium perenne ZmUbi_LpTC116908_nos expression cassette used in biolistic mediated transformation of Lolium perenne L.

FIGS. 17-28. SEQ ID NOs: 14, 15, 19, 20, 25, 23, 35, 38, 39, 40, and 42, respectively. S locus CDS variants. Detected sequence variation is identified within [ ] with both allelic forms described.

FIGS. 29-37. SEQ ID NOs: 52-54, 57-59, 62, 63 69 and 80, respectively. Z locus CDS variants. Detected sequence variation is identified within [ ] with both allelic forms described.

FIGS. 38-49. SEQ ID NOs: 80, 82, 85, 89, 90, 95, 96, 105, 108-110 and 112, respectively. Predicted Amino Acid translation showing S locus amino acid variants.

FIGS. 50-58. SEQ ID NOs: 122-124, 127-129, 132, 133 and 139, respectively. Predicted Amino Acid translation showing Z locus amino acid variants.

FIGS. 59-79. SEQ ID NOs: 141-161, respectively. Nucleic acid sequences of the ZmUbi_SI gene_nos expression cassettes used in biolistic mediated transformation of Lolium perenne L. Legend: Gateway attB1 site (bold underline); Zea mays Ubi promoter (italics)+intron (underlined italics); Lolium perenne coding region in antisense and sense orientations (underline); rga2 intron (bold); Nopaline synthase (nos) terminator (bold italics); Gateway attB2 site (bold underline)

FIG. 80. Pictorial description of the transformation pipeline; A, preparation of donor ryegrass material; B, somatic embryo callus initiation; C, callus proliferation; D, osmotic treatment; E, biolistic delivery of transgene including expression cassette; F, callus growth on tissue culture medium including appropriate selection agent; G, regeneration of putative transgenic plant from callus; H, establishment of putative transgenic plant.

FIG. 81. PCR evaluation of transgenic status for individual tillers from regenerated transgenic events. Each transformation event was assessed through three individual tillers split from the regenerated plant. Only examples where all three tillers gave positive confirmation of the presence of the transgene, did the event get accepted for further evaluation. The brackets with numbers 1 and 2 in the figure identify 1, a transgenic event that would be discarded as all tillers are negative for the presence of the transgene and 2, a transgenic event where all three tillers have generated a positive result for the presence of the transgene.

FIGS. 82 A and B. FDA staining of viable pollen grains for example transgenic plants with SiRNA constructs for the down regulation of a candidate S and Z gene respectively. Both viable and non-viable pollen grains can be seen in both A and B.

FIG. 83. Stages of ryegrass flower dissection. A—Intact flowers of ryegrass. Upon reaching reproductive maturity the anthers are released from the flower. The stigmatic papillae will then extend and become visible. B—individual spikelets were excised from a floral spike for further dissection. C—Both male and female reproductive tissues were excised from the spikelet. D and E—the female tissue was further excised to examine pollen tube growth on pollinated stigmas.

FIG. 84. Incompatible reaction of pollen tube growth. A and B, examples of single pollen grains germinating on stigmatic papillae and upon contact growth is arrested. The pollen tubes upon contact will often become swollen in shape through cytoplasmic pressure, indicated by arrows. C, An incompatible reaction of self pollination from a transgenic plant containing the SiRNA construct for LpOs05g0149600.

FIG. 85. Compatible pollen tube growth with untransformed plants. Pollen was taken from unrelated ryegrass plants and placed upon an untransformed flower. The pollen tube has made contact with the stigmatic papillae and has then continued to grow in a directed manner towards the ovary. The pollen tube upon growth will deposit callose plugs at regular intervals (indicated by arrows) to retain cytoplasmic pressure, allowing the sperm cells to successfully migrate towards the ovary. These vacated regions will become vacuolated.

FIG. 86. Compatible pollen reaction. The pollen tube has made contact with the stigmatic papillae and has then continued to grow in a directed manner towards the ovary. The compatible pollen tube will deposit callose plugs at regular intervals (indicated by arrows). The reaction was observed on self-pollination of a transgenic plant containing the siRNA construct for the LpOs06g0680500.

FIG. 87. Microscopic images of the pollen-stigma interaction of two different plants containing the siRNA construct for the LpOs05g0152900 gene. The two different transgenic events (A and B) show a range of phenotypes from incompatible to partially compatible.

FIGS. 88-108. Expression profiles of the Lolium perenne SI genes. Expression profiles were determined through BLAST analysis of sequence reads from multiple tissues of the Lolium perenne L. genotype Impact04, compared to the Brachypodium distachion CDS gene sequences used as orthologous templates.

FIG. 109. Schematic diagram of F1 hybrid grass breeding. Plants are initially genotyped using markers on or around the S and Z loci. Two parental pools are then generated and multiplied, with testing of the degree of heterosis between pools once sufficient seed has been generated.

EXAMPLES Example 1. Isolation of SI Genes

Both the S and Z locus was delimited through comparative genomics and BAC clone and genomic sequencing. All genes within the sequence data were identified (Tables 1 and 2). Sequences were determined through the FGENESH prediction software. Expression profiles were determined for each gene as described in Example 6. Expression profiles were determined through BLAST analysis of sequence reads from multiple tissues of the Lolium perenne L. genotype Impact04, compared to the Brachypodium distachion CDS gene sequences used as orthologous templates (See FIGS. 88-108).

TABLE 1 Genes identified within the S locus Nucleic Poly- acid peptide Ryegrass gene SEQ ID SEQ ID identified Syn Predicted Gene Function NO: NO: LpOs05g0481800 Protein prenyltransferase domain 1 71 containing protein, pentatricopeptide repeat- containing protein LpOs05g0147700 Cyclin-like F-box domain 2 72 containing protein LpOs05g0148300 Ribosomal protein S27, 3 73 mitochondrial family protein LpOs01g0254300 Similar to Pectinesterase-1 4 74 precursor (EC 3.1.1.11) (Pectin methylesterase 1) (PE 1) LpOs08g0226800 TRAF-like domain containing 5 75 protein, BTB/POZ and MATH domain-containing protein LpOs05g0148400 Conserved hypothetical protein 6 76 LpOs05g0148500 Electron transport accessory 7 77 protein domain containing protein LpOs07g0118800 Conserved hypothetical protein 8 78 LpOs07g0118900 Cyclin-like F-box domain 9 79 containing protein LpOs05g0148600 Na+/H+ antiporter 10 80 LpOs05g0148700 Armadillo-like helical domain 11 81 containing protein, senescence associated protein LpOs01g0372700 hypothetical protein, putative 12 82 asparagine--tRNA ligase, cytoplasmic 1-like LpOs05g0148900 Glutathione-S-transferase 19E50 13 83 LpOs01g0369700 Similar to Glutathione S- 14 84 transferase GST 8 LpOs05g0149100 C2 calcium/lipid-binding region, 15 85 CaLB domain containing protein LpOs05g0149200 PWWP domain containing 16 86 protein LpOs05g0149300 1-aminocyclopropane-1- 17 87 carboxylate oxidase LpOs05g0149400 1-aminocyclopropane-1- 18 88 carboxylic acid oxidase LpOs05g0149500 Lipopolysaccharide-modifying 19 89 protein family protein, predicted: O-glucosyltransferase rumi homolog LpOs05g0149600 Cullin-1 20 90 LpOs05g0149800 EF-Hand type domain containing 21 91 protein, serine/threonine-protein phosphatase 2A regulatory subunit B″ subunit gamma-like LpOs05g0149900 Tetratricopeptide-like helical 22 92 domain containing protein LpOs05g0150000 putative proline synthetase 23 93 associated protein LpOs05g0150300 probable chromatin-remodelling 24 94 complex ATPase chain-like protein LpOs05g0150400 Double-stranded RNA binding 25 95 domain containing protein LpOs05g0150500 Conserved hypothetical protein, 26 96 putative transport inhibitor response TIR1 LpOs05g0150600 ATP-dependent DNA helicase 27 97 RecQ family protein LpOs10g0545800 Cytochrome biosynthesis 28 98 CcmE/CycJ protein family protein LpOs05g0150700 Heavy metal 29 99 transport/detoxification protein domain containing protein LpOs05g0150800 Similar to Plastid 5,10- 30 100 methylene-tetrahydrofolate dehydrogenase, LpOs05g0150900 Histidyl-tRNA synthetase 31 101 LpOs02g0508100 hypothetical protein containing 32 102 DUF3339 LpOs05g0151000 Lpbcd762 Similar to RNA polymerase II 33 103 largest subunit LpOs05g0151100 Conserved hypothetical protein, 34 104 ferritin domain LpOs05g0151300 Rubber elongation factor family 35 105 protein LpOs05g0151400 Chloroplast protein import 36 106 component Toc86/159 family protein LpSb07g026730 Putative uncharacterized protein 37 107 LpOs05g0152400 Glycosyl transferase, family 14 38 108 protein, xylosyltransferase-like LpOs06g0680500 Glutamate receptor 3.4 precursor 39 109 (Ligand-gated ion channel 3.4) LpOs05g0152900 Seven in absentia protein family 40 110 protein LpOs05g0153000 Gelsolin family protein, villin-1- 41 111 like LpOs05g0153200 Region of unknown function, 42 112 putative Zinc finger, XS and XH domain containing protein LpOs05g0153300 Lipase, class 3 family protein 43 113 LpOs05g0153400 predicted pentatricopeptide 44 114 repeat-containing protein LpOs05g0153600 FAR1 domain containing protein 45 115 LpOs05g0154500 Spc97/Spc98 family protein, 46 116 gamma-tubulin complex LpOs05g0154600 Similar to VIP2 protein, 47 117 Hypothetical RING domain containing protein LpOs01g0652800 Protein of unknown function 48 118 DUF231, leaf senescence like protein, yellow leaf specific - like protein LpOs07g0286100 Cyclin-like F-box domain containing protein;

TABLE 2 Genes identified within the Z locus Nucleic Poly peptide Ryegrass gene acid SEQ SEQ ID identified Syn Predicted Gene Function ID NO: NO: LpOs04g0645100 LpTC101821 Tetratricopeptide-like 49 119 helical domain containing protein LpOs04g0645200 LpVQ VQ domain containing 50 120 protein LpOs07g0213300 pentatricopeptide repeat- 51 121 containing protein LpOs04g0645500 methyltransferase-like 52 122 protein 22-like LpOs04g0645600 Protein of unknown 53 123 function DUF6, transmembrane domain containing protein, vacuolar protein LpOs04g0647300 LpTC116908 Ubiquitin-specific protease 54 124 22 LpOs04g0647800 LpTC89057 Glycerol kinase 2 55 125 LpOs04g0647701 LpDUF247 Protein of unknown 56 126 function DUF247 LpOs03g0193400 Polyamine oxidase 57 127 precursor (EC1.5.3.11); LpOs06g0607800 Similar to 26S proteasome 58 128 regulatory complex subunit p42D LpOs06g0607900 C2 and GRAM domain 59 129 containing protein “No Pollen” LpOs11g0242400 Rieske [2Fe—2S] region 60 130 domain containing protein oxidoreductase LpOs04g0648400 Leucine rich repeat, N- 61 131 terminal domain containing protein LpOs04g0648500 BRCA1-associated 2 62 132 domain containing protein, Ubiquitin RING domain containing LpOs04g0648600 Conserved hypothetical 63 133 protein LpOs10g0419600 Chlorophyllase family 64 134 protein LpOs04g0648700 Conserved hypothetical 65 135 protein LpOs04g0274400 YL1 nuclear, C-terminal 66 136 domain containing protein LpOs04g0649200 Protein of unknown 67 137 function DUF869, filament-like plant protein 7-like LpOs04g0648800 RING-type domain 68 138 containing protein, zinc finger binding LpOs04g0648900 Dehydration responsive 69 139 element binding protein 2F, AP2 domain containing LpOs04g0649100 Pathogenesis-related 70 140 transcriptional factor and ERF domain containing protein, AP2 domain containing, Apetala 2 like LpOs04g0650000 Lpbcd266 Oryzain alpha chain precursor (EC 3.4.22.—)

Example 2. Resequencing Data Identified DNA Nucleotide Variance

A cohort of 21 genes were selected as key candidates of the S and Z loci. The genes were selected based on expression profile as well as sequence annotation.

The collection of 21 genes all had PCR primers designed to resequence the coding regions of the genes. The designed PCR amplicons were optimised to generate large genomic fragments. A total of 50 plant genotypes were used as the template DNA for resequencing. The 50 plants were chosen as a diverse spread of plants with a potential wide range of diversity to maximise allelic variation at the genic loci being resequenced.

The amplicons were generated, then pooled from each genotype and physically sheared to smaller fragments. DNA bar codes and sequencing adaptors were ligated onto the sheared fragments to identify each sample and then all samples were combined and sequenced using a next-generation Illumina MiSeq platform with 300 bp×2 reads.

The resulting sequence data was attributed back to the individual samples using the bar codes and was then checked for quality and low quality reads removed. The sequence reads were then reference aligned to the genes amplified and variant bases identified. The individual samples were then combined to give a dataset to identify all variant bases from the 50 samples, with potentially 100 different alleles.

The variant bases were recorded for each gene to identify if the variation was synonymous or non-synonymous in nature. A minimalistic requirement for each of the genes under investigation would be to have 5 or more variant amino acids identified within the transcript. A total of 5 variant amino acids would enable a maximum of 32 potential haplotypes from the data set allowing complete random mating maximal recombination.

As 100 haplotypes were resequenced, high levels of diversity are expected, however there could be a degree of overlap between the haplotypes from the plants chosen so the total number of unique haplotypes could be lower than the number sequenced.

Perennial ryegrass has been characterised as having a high degree of sequence variation within its genome, with estimates ranging from 1 SNP every 20-30 bases within a gene bases on resequencing 2-4 haplotypes. With two exceptions all of the genes resequenced contained sufficient variation in the coding regions of the genes that would generate a sufficient diversity of polypeptides that could deliver the required allelic variability (See FIGS. 17 to 58). Detected sequence variation is identified within [ ] with both allelic forms described.

The genes LpOs05g0151300 and LpOs05g0152400 did not have sufficient diversity, with only 3 and 2 variant amino acids respectively.

Example 3—Isolation of SI Genes: Cloning of the Ryegrass LpOs06g0607800 26S Proteasome Gene

In order to develop novel genetic markers for fine-scale genetic and physical mapping of the perennial ryegrass SI loci, linked heterologous cDNA-derived RFLP markers were selected for the S locus and Z on the basis of ortholocus co-segregation in cereal rye and/or blue canary grass. Molecular marker development, genetic mapping and region dissection is described in Shinozuka et al (2010). As a result of the assembled data sets fine-scale comparative sequence synteny with the model Poaceae species, specifically Oryza sativa and Brachypodium distachyon, was achieved for the delimited S and Z regions. Using the defined gene complement from the model Poaceae species, a BAC library was screened with primer pairs specific to the genes described and 39 specific clones were identified. The identity of the selected BAC clones was verified through direct sequencing of locus-specific amplicons. The specific BAC clones were then sequenced using Sanger and/or GSFLX technology and the resulting data was sequence assembled using the Newbler software package. Following sequencing and assembly gene-like nucleotide sequences were identified using BLAST and gene prediction software tools. Based on the derived information the reiteration of the procedure was performed for the selection of additional clones to further enhance the resolution and sequence data to assemble physical maps for the SI locus regions (FIGS. 1 and 2).

Molecular markers were developed from resequencing of specific genic loci, identified from the BAC sequencing and genetically mapped in a segregating population of Lolium perenne L. to confirm the location of the generated sequence.

The genome of a single Lolium perenne L. genotype (the plant—Impact04) has been sequenced to approximately 70× coverage, generating c. 2 billion sequencing reads of 100 by paired-end sequence reads on the Illumina GA2X and HiSeq2000 platform. The sequence data was filtered for high quality reads before being assembled using the SOAPdenovo v. 1.05 software package. The sequence assembly has been empirically optimised through iterative assessment of performance based on a range of input kmer sizes, in terms of number of bases assembled, and the average length of assembled contigs and scaffolds. An optimal assembly has generated 1.9 million scaffolds covering c. 1.7 Gb, while all contigs and singletons cover c. 3.5 Gb.

Comparison of contigs and scaffolds to the coding sequences of the model grass species Brachypodium distachyon L. permitted identification of putative perennial ryegrass orthologues to c. 86% of all predicted genes and alternate transcripts from the model grass species. A pipeline approach was implemented based on a highly parallel BLAST analysis method in order to group transcript and genomic sequences relevant to each individual Brachypodium gene sequence into individual local CAP3-based assemblies. This approach generated 23,285 genic files that were indexed to the corresponding Brachypodium gene. Development of the exome sequence library enabled identification of a large collection of genic contigs, along with the corresponding regulatory elements. The collection of contigs was then screened for the presence of the predicted genes within the S and Z loci that had not been identified through the BAC screening process.

A novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os06g0607800, hence the ryegrass gene was designated LpOs06g0607800 (SEQ ID NO: 58 and FIG. 3). The ryegrass gene was annotated as a 26S proteasome subunit gene through a BLASTx analysis (at e value=3e-65 compared to the rice amino acid sequence). The gene also contained an AAA ATPase domain (SEQ ID NO: 128).

In addition the ryegrass 26S proteasome subunit gene identified in the Z locus region through BAC sequencing, was compared through BLAST analysis to the genomic Impact04 sequence through BLAST analysis. The identification of the sequence from the BAC clones as well as the genomic sequence enabled the identification of variant sequence bases from the coding region of the gene (FIGS. 33 and 54).

Intracellular proteolysis is mainly regulated and enabled through the ubiquitin-proteasome pathway or the autophagy-lysozome/vacuole pathway. Proteolytic events play significant roles in SI through self-pollen rejection. Ubiquitin-mediated proteolysis is involved in the SI mechanism of the Brassicaceae and the Solanaceae.

The 26S proteasome consists of the 20S core proteasome (CP) element and the 19S regulatory particle (RP). Proteolysis occurs in the 20S compartment, while the 19S element confers ATP dependence and substrate specificity to the CP. The RP consists of two elements: a ring of six AAA-ATPase subunits (often abbreviated as RPT) that is expected to function in target unfolding and transport, and three non-ATPase subunits (often abbreviated as RPN). As the 26S Proteasome subunit gene LpOs06g0607800 contains the AAA-ATPase domain, it is of the class RPT.

Arabidopsis thaliana L. mutant lines, in which the RTP2 subunit of the 26S proteasome gene is disrupted, have demonstrated that male and female gamete transmission require a normal copy of the RPT2 gene to avoid abortion and failure in gametogenesis. In tobacco (Nicotiana tabacum L.) the NtRpn3 gene was found to physically interact with a calcium-dependent protein kinase and become phosphorylated in a calcium dependent manner.

While applicants do not wish to be restricted by theory, the LpOs06g0607800 26S Proteasome gene is hence proposed to be the female determinant of the Z locus.

Example 4—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs06g0607800 26S Proteasome Gene

The LpOs06g0607800 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 500 bp of coding sequence of the LpOs06g0607800 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).

The selection cassette was synthesized by a commercial gene synthesis vendor (GeneArt, Life Technologies) and cloned into a Gateway-enabled vector. The LpOs06g0607800 expression cassette was synthesized by a commercial gene synthesis vendor (GeneArt, Life Technologies) with flanking attB sites. For delivery in cis the LpOs06g0607800 expression cassette was sub-cloned into pDONR221 II (Invitrogen, Life Technologies) in a BP Clonase reaction. The resulting ENTRY clone was used in a LR Clonase II (Invitrogen, Life Technologies) reaction with the Gateway-enabled vector encoding the hph expression cassette. Colonies of all assembled plasmids were initially screened by restriction digestion of miniprep DNA. Restriction endonucleases were obtained from New England BioLabs (NEB; Ipswich, Mass.) and Promega (Promega Corporation, WI). Plasmid preparations were performed using the QlAprep Spin Miniprep Kit (Qiagen, Hilden) or the Pure Yield Plasmid Maxiprep System (Promega Corporation, WI) following the instructions of the suppliers. Plasmid DNA of selected clones was sequenced using ABI Sanger Sequencing and Big Dye Terminator v3.1 cycle sequencing protocol (Applied Biosystems, Life Technologies). Sequence data were assembled and analyzed using the SEQUENCHER™ software (Gene Codes Corporation, Ann Arbor, Mich.).

An ideogram of the gene expression cassette is shown in FIG. 4. The full sequence of the expression cassette is shown in FIG. 75.

Example 5—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs06g0607800 26S Proteasome Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs06g0607800 26S Proteasome gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass.

The vector used along with the transformation protocol has previously been used successfully in plant transformation experiments (Bilang, et al., 1991; Spangenberg, et al., 1995a; Spangenberg, et al., 1995b; Ye, et al., 1997; Bai, et al., 2001). The perennial ryegrass biolistic transformation method is outlined in FIG. 80.

Phenotypic evaluation of the resulting transgenic plants was performed by growing the plants to maturity, then following a vernalisation period of at 5° C. with 12 hour lighting for ten weeks, the plants were subjected to 22° C. with 24 hour lighting. The change in temperature and perceived day length initiated flowering in the plants, which then occurred 2-3 weeks later. Before the ryegrass flowers opened the flowering spike was checked for morphological alterations or deformities and was then contained within a paper bag to isolate the flowers from other potential pollen donors. The flowering spike may be maintained in its isolated state until flowering is complete, at which time seed set may be assessed. Multiple spikes may be bagged per plant and each spikelet and flower assessed visually for seed production.

Once a putative transgenic plant had regenerated on the selective medium, the plant was split into three single plant tillers. Each tiller was individually screened for the presence of the transgene through conventional PCR. Oligonucleotide primers had been designed to the promoter region that originated from the Ubiquitin (Ubi) gene from Zea mays as well as a second assay that targeted the RGA2 intron sequence from Triticum turgidum subsp, which interrupts the inverted ryegrass repeat of the target gene. These regions were chosen to minimise cross amplification from the endogenous ryegrass genome. The assays were developed and initially tested on untransformed ryegrass genomic DNA to confirm that cross amplification did not occur. Transgenic events were only selected when all three tillers returned a positive result for the presence of the transgene with both assays. If variable results were seen, the single tillers that were positive were returned to growth medium and grown further, until they could be resplit into three tillers and screened again.

FIG. 81 shows PCR evaluation of transgenic status for individual tillers from regenerated transgenic events. Each transformation event was assessed through three individual tillers split from the regenerated plant. Only examples where all three tillers gave positive confirmation of the presence of the transgene, did the event get accepted for further evaluation. The brackets with numbers 1 and 2 in the figure identify 1, a transgenic event that would be discarded as all tillers are negative for the presence of the transgene and 2, a transgenic event where all three tillers have generated a positive result for the presence of the transgene.

An average of 12 different transgenic events per construct were generated, with a range of 3-19. The plants were transferred to soil and were maintained in an appropriate containment glasshouse.

TABLE 3 Numbers of transgenic events generated for the seven candidate genes undergoing functional evaluation. Positive Transgenic Gene Number Gene Name Events Generated LpOs04g0648500 Ubiquitin-specific protease 22 3 LpOs05g0149600 Cullin 15 LpOs06g0607800 26S proteasome 8 LpOs06g0680500 Glutamate receptor 11 LpOs06g0607900 NoPollen Gram domain 11 LpOs05g0152900 SIAHa 18 LpOs04g0647300 TC116908 19

Phenotypic evaluation was performed upon the plant reaching reproductive maturity. Initially pollen grains were assessed for viability to ensure a response would be seen. A common effect on plant cells passing through a transformation and tissue culture process is reduced fertility of the resultant whole plant. The viability was confirmed through staining with fluorescein diacetate (FDA). FDA is a lipophilic compound and is membrane-permeable and non-fluorescent. Viable pollen grains will have intracellular esterase activity and will be able to perform enzymatic hydrolysis of FDA upon its entry into the cell. Once FDA has been hydrolyzed within the viable pollen grain it will be a highly fluorescent compound that is unable to diffuse out of the cell and will be retained, producing an intense green fluorescence within the cytoplasm.

FIG. 82 shows FDA staining of viable pollen grains for example transgenic plants with SiRNA constructs for the down regulation of a candidate S and Z gene respectively. Both viable and non-viable pollen grains can be seen in both A and B.

Once pollen viability was confirmed pollen-pistil interactions were assessed. Dissection of the floral tissues of flowering ryegrass plants were performed to microscopically assess pollen-pistil interactions.

FIG. 83 shows stages of ryegrass flower dissection. A—Intact flowers of ryegrass. Upon reaching reproductive maturity the anthers are released from the flower. The stigmatic papillae will then extend and become visible. B—individual spikelets were excised from a floral spike for further dissection. C—Both male and female reproductive tissues were excised from the spikelet. D and E—the female tissue was further excised to examine pollen tube growth on pollinated stigmas.

Each transgenic event was represented by three plants as described in the PCR screening process. Multiple transgenic events are required as the insertion of the transgene SiRNA construct is likely to result in a range of expression levels. This difference in expression between the transgenic events is likely to lead to a range of phenotypes for the reaction. The pollen-pistil compatible/incompatible reaction can be visualised through pollen tube abortion upon contact with the stigmatic tissue, or pollen tube directed growth towards the ovary. Multiple flowers per plant are required to be assessed for confidence over the observed phenotype. Once pollinated stigmatic tissues were isolated, aniline blue staining was performed and the tissue visualised under an inverted fluorescent microscope. A range of reactions were observed. Incidences of self-incompatibility was seen for many plants, while instances of partial compatibility was also seen.

FIG. 84 shows an incompatible reaction of pollen tube growth. A and B, examples of single pollen grains germinating on stigmatic papillae and upon contact growth is arrested. The pollen tubes upon contact will often become swollen in shape through cytoplasmic pressure, indicated by arrows. C, An incompatible reaction of self pollination from a transgenic plant containing the SiRNA construct for LpOs05g0149600.

FIG. 85 shows a compatible pollen tube growth with untransformed plants. Pollen was taken from unrelated ryegrass plants and placed upon an untransformed flower. The pollen tube has made contact with the stigmatic papillae and has then continued to grow in a directed manner towards the ovary. The pollen tube upon growth will deposit callose plugs at regular intervals (indicated by arrows) to retain cytoplasmic pressure, allowing the sperm cells to successfully migrate towards the ovary. These vacated regions will become vacuolated.

FIG. 86 shows a compatible pollen reaction. The pollen tube has made contact with the stigmatic papillae and has then continued to grow in a directed manner towards the ovary. The compatible pollen tube will deposit callose plugs at regular intervals (indicated by arrows). The reaction was observed on self-pollination of a transgenic plant containing the siRNA construct for the LpOs06g0680500.

FIG. 87 shows microscopic images of the pollen-stigma interaction of two different plants containing the siRNA construct for the LpOs05g0152900 gene. The two different transgenic events (A and B) show a range of phenotypes from incompatible to partially compatible.

Example 6—Expression Analysis of the LpOs06g0607800 26S Proteasome Gene

A single genotype of perennial ryegrass was subjected to transcriptome analysis through deep-sequencing of cDNA samples derived from multiple distinct tissue types. A total of 19 different RNA samples were generated from vegetative tissues, including leaf, pseudostem and root samples for both terrestrial and subterranean aspects of gene expression (Table 4). In addition a collection of reproductive libraries were generated from anthers, pistils, stigmas and pollinated pistils. The libraries were prepared for Illumina-based sequencing using the RNASeq preparation method. Each library was internally bar-coded to permit discrimination following the sequencing process.

A total of c. 0.6 billion sequencing reads were generated from the Illumina HiSeq2000 platform. Approximately 30 million sequence reads was generated from each tissue sample. The generated sequences were then filtered and quality trimmed to ensure <3 bases per sequence read were called as “N” and mean and local Phred quality was >30 in all instances.

The quality filtered reads were then BLASTn analysed against the coding sequences of the Brachypodium distachion genome. The number of BLASTn matches per gene were counted per tissue type and tabulated. As the number of reads generated per sample varied, the BLASTn mapped read count was normailsed on the 75^(th) percentile to generate normalised values for comparative analysis. The Brachypodium distachion genome was used as a whole genome reference in this analysis to mitigate issues of gene absence or incomplete assemblies of any de novo generated gene catalogue.

TABLE 4 Gene expression analysis through RNA sequencing from different tissues of Lolium perenne L. Number of unique reads aligned Tissue name - to the Brachypodium CDS gene library Description of tissue source catalogue Tip 1 Tip of the youngest leaf from a single tiller 22,752,873 Tip 2 Tip of the second youngest leaf from a single 18,298,951 tiller Tip 3 Tip of the third youngest leaf from a single tiller 14,929,714 Mid 1 Mid section of the youngest leaf from a single 17,729,494 tiller Mid 2 Mid section of the second youngest leaf from a 18,952,545 single tiller Mid 3 Mid section of the third youngest leaf from a 13,934,280 single tiller Pseudo 1 Complete pseudostem from a single tiller 9,887,218 Pseudo 2 Lower portion of the pseudostem of a single 12,245,297 tiller Pseudo 3 Upper portion of the pseudostem of a single 12,594,959 tiller Root Mid Mid section of root mass 12,571,148 Root Tip Tip section of root mass 12,239,578 Flower Complete flower, (un)opened 9,920,235 Pollinated Pistil Self pollen added to pistil, then after 5 minutes 10,840,818 5 mins pistil excised and frozen Pollinated Pistil Self pollen added to pistil, then after c. 1 hour 9,281,790 1 hour minus pistil excised and frozen Pollinated Pistil Self pollen already added to pistil, upon tissue 11,208,524 1 hour plus harvest, for an undefined time greater than 1 hour prior to pistil being excised and frozen Stigma Harvested following pollination at 0 minutes 9,724,120 pollinated 0 mins Stigma Harvested following pollination at 5 minutes 13,258,777 pollinated 5 mins Pistil Complete pistil, without pollen 13,714,389 Anther Complete anther without(out) pollen grains 13,724,305

The nucleic acid sequence identified as the LpOs06g0607800 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi1g36400 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a dramatic increase in gene expression over time in the pistil tissues upon self pollination (FIG. 104). A constant level of gene expression is detected across the entire plant, however some interference in the analysis from other gene family members is possible. Alternatively, the generic expression of the gene may have an alternative function across all tissues, or may demonstrate non-tissue specific gene expression as a result of particular promoter elements.

Example 7—Isolation of SI Genes: Cloning of the Ryegrass LpOs05g0149600 Cullin Gene

Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os05g0149600, hence the ryegrass gene was designated LpOs05g0149600 (SEQ ID NO: 20 and FIG. 5). The ryegrass gene was annotated as a Cullin gene through BLASTx analysis (SEQ ID NO: 90).

In addition the ryegrass Cullin gene identified in the S locus region through BAC sequencing, was compared through BLAST analysis to the genomic Impact04 sequence through BLAST analysis. The identification of the sequence from the BAC clones as well as the genomic sequence enabled the identification of variant sequence bases from the coding region of the gene (FIG. 21).

Cullins are molecular scaffolds responsible for assembling RING-based E3 ubiquitin ligases. Within the Solanaceae, Rosaceae and Plantaginaceae families the SI mechanism involves the formation of a complex consisting of a Cullin gene, an F-box gene along with a suppressor of kinetochore protein. The complex possesses the ubiquitin E3 ligase activity that attaches polyubiquitin chains to target proteins, such that ubiquitinated proteins are degraded by the 26S proteasome. The Cullin gene within the complex plays a role in assembling the other sub-units, and links to a further compound that recruits ubiquitin proteins to attach to the target proteins.

Example 8—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs05g0149600 Gene

The LpOs05g0149600 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 500 bp of coding sequence of the LpOs05g0149600 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).

The selection cassette was synthesized, delivered and sequenced as described in Example 4.

An ideogram of the gene expression cassette is shown in FIG. 6. The full sequence of the expression cassette is shown in FIG. 63.

Example 9—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs05g0149600 Cullin Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs05g0149600 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.

Example 10—Expression Analysis of LpOs05g0149600 Cullin Gene

Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs05g0149600 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi2g35830 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a constitutive level of gene expression in all tissues (FIG. 92). However, significantly elevated levels of gene expression are seen in pistil and pollinated pistils at 5 minutes as well as whole flower and stigma at 0 and 5 minutes. The pattern of expression seen can be described as increasing in stigma from 0 to 5 minutes along with a corresponding increase in pistil at 5 minutes that then decreases to the constitutive level at a 1 hour time point.

Example 11—Isolation of SI Genes: Cloning of the Ryegrass LpOs06g0680500 Glutamate Receptor Gene

Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC-clone related sequence that displayed sequence similarity with the rice gene Os06g0680500, hence the ryegrass gene was designated LpOs06g0680500 (FIG. 7 and SEQ ID NO:39). The ryegrass gene was annotated as a glutamate receptor gene through a BLASTx analysis (e value=0 compared to the rice amino acid sequence) and was identified as containing the requisite GABA domain (SEQ ID NO: 109).

Glutamate receptor genes have been identified in Arabidopsis thaliana and tobacco as forming influx channels in tip cell types that undergo directed patterns of growth such as those of pollen tubes, as well as root hairs. Studies on Arabidopsis root cells show that glutamate induces a sharp depolarization of the membrane potential, and a concomitant rise in intracellular calcium. Growth of tobacco pollen tubes in the presence of a glutamate receptor antagonist has been shown to be repressed, as is also the case for specific directed uptake of calcium. Gene knock-out experiments of pollen expressing glutamate receptor genes in Arabidopsis have documented reduction in growth rates as well as abnormal morphology of the tip and tube.

While applicants do not wish to be restricted by theory, LpOs06g0680500 LpGlu1 Glutamate receptor gene is hence proposed to be the male determinant of the S locus.

Example 12—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs06g0680500 LpGlu1 Gene

The nucleic acid sequence identified as LpOs06g0680500 gene has a 484 bp fragment selected as a design element for expression cassette. The Zea mays ubiquitin gene promoter (Christensen et al. 1992) was used to drive expression and the nopaline synthase (nos) gene terminator (Bevan, 1984; Rogers et al., 1985) was selected to arrest transcription.

The LpGlu1 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 484 bp of coding sequence of the LpGlu1 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).

The selection cassette was synthesized, delivered and sequenced as described in Example 4.

An ideogram of the gene expression cassette is shown in FIG. 8. The full sequence of the expression cassette is shown in FIG. 68.

Example 13—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs06g0680500 Glutamate Receptor Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs06g0680500 LpGlu1 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.

Example 14—Expression Analysis of the LpOs06g0680500 Glutamate Receptor Gene

Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs06g0680500 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi1g32800 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies high levels of gene expression in vegetative tissues and some gene expression in anthers (FIG. 97). The glutamate genes represent a gene family within the Brachypodium distachion genome that will be involved in many functions across the different plant tissues and consequently mapping of related sequences or alternative functions for glutamate genes is likely and could explain the constitutive expression. Nevertheless, a significant increase in expression is detected in anthers and not in the female pistil or stigma tissues.

Example 15—Isolation of SI Genes: Cloning of the Ryegrass LpOs04g0648500 Gene

Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os04g0648500, hence the ryegrass gene was designated LpOs04g0648500 (See FIG. 9 and SEQ ID NO: 62). The perennial ryegrass gene was identified as physically linked to the TC116908-related gene. The TC116908-derived genetic marker co-segregated with the Z locus in rye (Secale cereale L.) (Hackauf and Wehling 2005). In the perennial ryegrass BAC clone including the TC116908 orthologue, LpOs04g0648500 and other 2 genes were identified (Shinozuka et al. 2010).

The LpOs04g0648500 gene was annotated as an Ubiquitin-specific protease 22 gene through a BLASTx analysis (SEQ ID NO: 132). The gene contained a Znf-UBP and BRAP2 domains. The Znf-UBP domain exhibits the ubiquitin-specific protease activity and functions as protein stabiliser through target-specific de-ubiquitinylation. The human BRAP2 domain was originally identified as interacting with the BRCA1 (breast cancer 1) gene products. A sequence homology search indicated that this domain is also conserved in the Arabidopsis At2g26000 [zinc finger (ubiquitin-hydrolase) domain-containing protein] and At2g42160 [zinc finger (C3HC4-type RING finger) family protein] gene products, suggesting that this gene is involved in the ubiquitin-proteasome system.

In the S-RNase-based and Brassicaceae-type SI systems, involvement of the ubiquitin-proteasome system has been suggested. The 26S proteasome complex is bound with a UBP, and the 19S regulatory particle of the 26S proteasome complex is activated by the UBP. In the Z locus-linked BAC clones, 26S proteasome-related genes (LpTC116908 and LpOs06g0607800) were identified, of which products may interact with the LpOs04g0648500 gene product.

While applicants do not wish to be restricted by theory, the LpOs04g0648500 Ubiquitin-specific protease 22 gene is hence proposed to be one of the SI determinants in the Z locus

Example 16—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs04g0648500 Gene

The nucleic acid sequence identified as LpOs04g0648500 gene has a 578 bp fragment selected as a design element for expression cassette. The Zea mays ubiquitin gene promoter (Christensen et al. 1992) was used to drive expression and the nopaline synthase (nos) gene terminator with (Bevan, 1984; Rogers et al., 1985) was selected to arrest transcription.

The LpOs04g0648500 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 578 bp of coding sequence of the LpOs04g0648500 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).

The selection cassette was synthesized, delivered and sequenced as described in Example 4.

An ideogram of the gene expression cassette is shown in FIG. 10. The full sequence of the expression cassette is shown in FIG. 77.

Example 17—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs04g0648500 Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs04g0648500 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.

Example 18—Expression Analysis of the LpOs04g0648500 Gene

Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs04g0648500 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi5g23970 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a low level of constitutive expression in all tissues, with an increase in all of the reproductive samples. The highest level of gene expression was detected in the pistil samples (See FIG. 106).

Example 19—Isolation of SI Genes: Cloning of the Ryegrass LpOs06g0607900 Gene

Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os06g0607900, and the ryegrass gene was hence designated LpOs06g0607900 (See FIG. 11 and SEQ ID NO: 59). The gene contained both the C2 and GRAM amino acid domains (SEQ ID NO: 129). The C2 domain is comprised of 2 highly conserved domains that are separated by a basic region. The C2 domain is a calcium-dependent membrane-targeting module that is found in proteins involved in signal transduction or membrane trafficking. The domain is often involved in calcium-dependent phospholipid binding and in membrane targeting processes. The GRAM domain is a glucosyltransferase, Rab-like GTPase activators and myotubularin domain. The domain is associated with membrane-coupled processes and signal transduction. The GRAM domain was first computationally identified in 2000 (Doerks et al.) and functional analysis of the domain has since elucidated roles in protein association with a target membrane.

The rice homologue of the novel gene identified from the BAC sequence characterisation has been partially described in its function and has been designated the “no-pollen” gene (Osnop). The rice Osnop gene was identified and characterised through a Ds transposon insertion strategy. The deleted gene displayed abnormal anthers and no pollen production. Through promoter fusions with the GUS reporter gene, the endogenous gene was characterised as showing gene expression late in pollen formation and in the germination of pollen tubes (Jiang et al. 2005).

While applicants do not wish to be restricted by theory, the LpOs06g0607900 No-Pollen (LpNOP) gene is hence proposed to be the male determinant of the Z locus.

Example 20—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs06g0607900 LpNOP1 Gene

The LpOs06g0607900 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 400 bp of coding sequence of the LpOs06g0607900 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).

The selection cassette was synthesized, delivered and sequenced as described in Example 4.

An ideogram of the gene expression cassette is shown in FIG. 12. The full sequence of the expression cassette is shown in FIG. 76.

Example 21—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs06g0607900 Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs06g0607900 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.

Example 22—Expression Analysis of the LpOs06g0607900 Gene

Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs06g0607900 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi1g36390 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies high levels of gene expression almost exclusively in anthers. Limited expression has been detected in flowers and stigma pollinated at 0 minutes, which could result from anthers in the flower or initial germination of the pollen grain (See FIG. 105).

Example 23—Isolation of SI Genes: Cloning of the Ryegrass LpOs05g0152900 a Seven-in-Absentia Homologue Gene

Using the methods outlined in Example 3, from the exome sequencing of the genotype Impact04, a Lolium perenne L. gene was identified that displayed sequence similarity with the rice gene Os05g0152900 (FIG. 13 and SEQ ID NO: 40). Due to sequence similarity with seven in absentia homologue (SIAH) genes, the identified gene was designated LpSIAH (SEQ ID NO: 110).

SIAH proteins consist of a RING finger domain at the N-terminus and a Sina domain at the C-terminus, and have an ubiquitin-E3 ligase activity when a homodimer is formed (Den Herder et al. 2008). Suppression of SIAH protein function in plant species results in increased root systems, enlarged leaves and increased shoot number, suggesting that SIAH protein is involved in a wide range of plant developmental processes.

Substrates of SIAH proteins are degraded in an ubiquitin-related pathway following interaction. Glutamate receptor proteins are substrates of SIAH proteins. The RING finger domain of SIAH protein and the Siah-interacting domain of glutamate receptor proteins are essential for interaction. Interaction of the SIAH and glutamate receptor proteins exerts effects on calcium current modulation. A glutamate receptor-like gene, LpGlu1, was identified as being located physically close to LpSIAH.

Example 24—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpOs05g0152900 LpSIAH Gene

The LpSIAH expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 400 bp of coding sequence of the LpSIAH gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault and Melcher 1993).

The selection cassette was synthesized, delivered and sequenced as described in Example 4.

An ideogram of the gene expression cassette is shown in FIG. 14. The full sequence of the expression cassette is shown in FIG. 69.

Example 25—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpOs05g0152900 LpSIAH Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpOs05g0152900 LpSIAH gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.

Example 26—Expression Analysis of the LpOs05g0152900 LpSIAH Gene

Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpOs05g0152900 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi2g35550 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies gene expression predominantly in anthers and the pollinated stigma at 0 minutes. Low or negligible expression in all other tissues was observed (See FIG. 98).

Example 27—Isolation of SI Genes: Cloning of the Ryegrass LpTC116908 Gene

Using the methods outlined in Example 3, a novel Lolium perenne L. gene was identified from BAC clone-related sequence that displayed sequence similarity with the rice gene Os04g0647300 and the barley gene TC116908, hence the ryegrass gene was designated LpTC116908. In rye (Secale cereale L.), a TC116908 derived genetic marker co-segregated with the rye Z locus. Close to the TC116908 derived marker, 4 genetic markers were located (Hackauf and Wehling 2005). The corresponding genetic markers were assigned to the lower part of LG2 of perennial ryegrass (Shinozuka et al. 2010). BAC clones containing the genetic marker-related sequences were sequenced to identify Lolium perenne L. genes encoded in the Z locus (See FIG. 15 and SEQ ID NO: 54).

The LpTC116908 gene was annotated as an Ubiquitin-specific protease 22 gene through a BLASTx analysis (SEQ ID NO: 124). The gene contained a Znf-UBP (Zinc finger ubiquitin-specific processing protease) and peptidase C19 domains. The Znf-UBP domain exhibits the ubiquitin-specific protease activity and functions as protein stabiliser through target-specific de-ubiquitinylation. The peptidase C19 domain shares sequence similarity to the Znf-UBP-like domain and possesses ubiquitin-specific peptidase activity. The LpTC116908 gene was expressed in perennial ryegrass reproduction organs (Shinozuka et al. 2010).

In the S-RNase-based and Brassicaceae-type SI systems, involvement of the ubiquitin-proteasome system has been suggested. The 26S proteasome complex is bound with a UBP, and the 19S regulatory particle of the 26S proteasome complex is activated by the UBP. In the Z locus-linked BAC clones, 26S proteasome-related genes (LpOs06g0607800 and LpOs04g0648800) were identified, of which products may interact with the LpTC116908 gene product.

While applicants do not wish to be restricted by theory, the LpTC116908 Ubiquitin-specific protease 22 gene is hence proposed to be one of SI determinants of the Z locus

Example 28—Generation of Transformation Vectors Containing an Inverted Hairpin Structure of the LpTC116908 LpOs04g0647300 Gene

The LpTC116908 expression cassette consists of the promoter, 5′ untranslated region and intron from the Ubiquitin (Ubi) gene from Zea mays (Toki et al 1992) followed by 492 bp of coding sequence of the LpTC116908 gene from L. Perenne in an inverted repeat interrupted by intron 2 of the RGA2 gene from Triticum turgidum subsp. durum (Douchkov et al 2005). The hairpin cassette was terminated with the 3′ untranslated region (UTR) comprising the transcriptional terminator and polyadenylation site of the nopaline synthase gene (nos) from A. tumefaciens pTi15955 (Fraley et al 1983).

The selection cassette (delivered either in cis or trans) comprised of the promoter, 5′ untranslated region and intron from the Actin (Act1) gene from Oryza sativa (McElroy et al 1990) followed by a synthetic, version of hph gene from E. coli (Kaster et al 1983) codon-optimized for expression in monocots, which encodes a protein that confers resistance to the antibiotic hygromycin. This cassette was terminated with the 3′ UTR comprising the transcriptional terminator and polyadenylation sites from the 35s gene of cauliflower mosaic virus (CaMV) (Chenault et al 1993).

The selection cassette was synthesized, delivered and sequenced as described in Example 4.

An ideogram of the gene expression cassette is shown in FIG. 16. The full sequence of the expression cassette is shown in FIG. 73.

Example 29—Biolistic Transformation of Perennial Ryegrass (Lolium perenne) for Expression of dsRNA Products of the LpTC116908 Gene for RNAi-Mediated Down-Regulation of SI

Biolistic co-transformation of perennial ryegrass with the vectors containing the LpTC116908 gene sequence, driving the expression of the RNAi cassette and the synthetic version of hph gene from E. coli for hygromycin resistance was conducted on embryogenic calli for perennial ryegrass, as described in Example 5.

Example 30—Expression Analysis of the LpTC116908 Gene

Using the methods outlined in Example 6, the nucleic acid sequence identified as the LpTC116908 gene, when compared against the coding portion of the Brachypodium distachion genome sequence, identifies Bradi5g23920 as the closest matching gene sequence. The expression profile that has been generated from this analysis identifies a low level of constitutive expression in all tissues, with a significant increase in both of the pollinated stigma samples. Slight increases in gene expression are also identified in pistil samples as well as pseudostems (FIG. 102).

Example 31. Generation of Transformation Vectors Containing an Inverted Hairpin Structure

SiRNA constructs were prepared for all 21 candidate genes, using the methods outlined in Example 4. From the resequencing data the most conserved 300-500 bp region of the gene was chosen for the design.

S Locus

LpOs05g0148600

LpOs01g0369700

LpOs05g0149100

LpOs05g0149500

LpOs05g0149600

LpOs05g0150400

LpOs05g0150500

LpOs05g0151300

LpOs05g0152400

LpOs06g0680500

LpOs05g0152900

LpOs05g0153200

Z Locus

LpOs04g0645500

LpOs04g0645600

LpOs04g0647300

LpOs03g0193400

LpOs06g0607800

LpOs06g0607900

LpOs04g0648500

LpOs04g0648600

LpOs04g0648900

FIGS. 59 to 79 show nucleic acid sequence of the expression cassettes used in biolistic mediated transformation of Lolium perenne L. Legend: Gateway attB1 site (bold underline); Zea mays Ubi promoter (italics)+intron (underlined italics); Lolium perenne coding region in antisense and sense orientations (underline); rga2 intron (bold); Nopaline synthase (nos) terminator (bold italics); Gateway attB2 site (bold underline)

Example 32. Application of Genomic Data from the S and Z Interval in F1 Hybrid Grass Breeding

Heterosis or hybrid vigour is the phenomenon where the performance of an F₁ hybrid is greater than that of the parents. Ryegrass cultivars today are commonly bred from a limited number of elite parents (4-12), polycrossed together and then further polycrossed to bulk up seed numbers suitable for commercial sale. There are no commercial activities or schemes to capture heterosis in ryegrass breeding currently.

The identification of genetic markers in linkage disequilibrium with S and Z loci enables haplotypic prediction. The ability to genotype individuals for S and Z haplotypes opens a new avenue for efficient F₁ hybrid ryegrass production by selectively bottlenecking and combining haplotypes. The production of F₁ hybrid ryegrass by selectively bottlenecking SI alleles using linked genetic markers displays the greatest potential for cost effective application to commercial ryegrass breeding in the near future. The application of SI genetic markers to bottleneck SI allows breeders to work with any germplasm at their disposal with no prior requirements. The haplotype defining SI markers will enable breeders to selectively bottleneck SI haplotypes (without selfing) within defined pools to reduce the within pool compatibility and then bring two pools together for random crossing, where the SI haplotypes ensure between pool compatibility is greater than within, resulting in an increased production of F₁ progeny.

For example, in the initial stages a breeding nursery of phenotypically elite plants would be genotyped with the SI linked molecular markers and haplotypic prediction would be performed. Pairs of individuals (termed parent pools) would then be identified where one individual is heterozygous at both S and Z and the other individual is homozygous at one locus, either S or Z, and heterozygous at the other locus (for the same haplotypes present in the heterozygous individual), for example:

Ind x s1s2-z1z2

Ind y s1s2-z1z1

Two parent pools where the S and Z haplotypes between pools are completely different are identified and taken forward to the next step. The two selected pools are referred to as pool A and B in the follow stages.

Following from the initial parental pool development the seeds are multiplied. During this seed bulk stage, pool A and B are maintained in isolation to ensure no foreign SI haplotypes are introduced through pollen flow or external seed.

One round of random mating within each pool will bring the S and Z haplotype frequencies to equilibrium, with 50% of individuals heterozygous at both the S and Z loci, 25% homozygous at S or Z for one of the haplotypes, and the remaining 25% of individuals homozygous at the same locus but for the opposing haplotype, for example:

25% inds—s1s1-z1z2

50% inds—s1 s2-z1z2

25% inds—s2s2-z1z2

Continued unselected random mating within the two pools will maintain the homozygous and heterozygous frequencies whilst increasing seed numbers. In every round of mating, the heterozygous locus will alternate, for example:

25% - s1s1-z1z2 25% - s1s2-z1z1 25% - s1s1-z1z2 50% - s1s2-z1z2 → 50% - s1s2-z1z2 → 50% - s1s2-z1z2 25% - s2s2-z1z2 25% - s1s2-z2z2 25% - s2s2-z1z2

Pollen within pools will never be compatible with individuals heterozygous at both the S and Z loci. Consequently those individuals (which make up 50% of the plants) will only be pollen donors, not producing any seed, resulting in a 50% seed production rate within pools.

Once sufficient seed has been generated within pools, equal numbers of seed would be combined and sown out for F₁ seed production. As pollen from within pools is not compatible with the respective S and Z heterozygous individuals, only pollen from between the two pools will fertilize those individuals, resulting in those plants yielding 100% F₁ hybrid seed. The remaining individuals, which are compatible with pollen both from within and between pools will yield both F₁ hybrid and within-pool seed. However, as there is a greater number of compatible haplotypic combinations from between-pools, than from within-pools, a higher proportion of the seed will be F₁ hybrids.

From simulation of all the haplotypic combinations between and within-pools, and the proportion of compatible combinations, the theoretical percentage of F₁ hybrid seed produced following the described scheme is >83%. With hybrid individuals likely to be more vigorous and competitive than within-pool seed, the proportion of hybrids, >83%, is likely to increase on farm when grown under a competitive sward situation. In the proposed breeding design, pool A and B will reach haplotype frequency equilibrium after one round of crossing, meaning that breeders can bulk seed up within pools over as many generations as deemed necessary, as long as the pools are maintained in isolation from foreign pollen. This also allows breeders to perform small test crosses between pools with each seed bulk up to ensure heterosis still remains in the progeny. At no point during the breeding design is there a requirement for controlled pollination.

This breeding design could be applied to any outbreeding grass species belonging to the Poaceae that has the S and Z loci regulating self-incompatibility without limitation. More preferably the grass species would be of the Bambusoideae, Ehrhartoideae (formerly Oryzoideae) or Pooideae Glade. More preferably the grass species would be of the tribe Poeae. More preferably the grass species would be of the genera Lolium, Festuca, Poa, Dactylis, Bromus, Secale, Pennisetum and Panicum. More preferably the grass species would be of the genera Lolium and Festuca. More preferably the species would be of the genus Lolium. More preferably the Lolium species would be Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass), Lolium boucheanum (hybrid ryegrass) Lolium arundinaceum (tall fescue) and Lolium pratense (meadow fescue).

FIG. 109 shows a schematic diagram of F1 hybrid grass breeding. Plants are initially genotyped using markers on or around the S and Z loci. Two parental pools are then generated and multiplied, with testing of the degree of heterosis between pools once sufficient seed has been generated.

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The claims defining the invention are as follows:
 1. A method for controlling hybridization in a plant, said method including: establishing or identifying a first plant strain with a first Z locus haplotype and a first S locus haplotype; establishing or identifying a second plant strain with a second Z locus haplotype different from the first Z locus haplotype and a second S locus haplotype different from the first S locus haplotype; and crossing said plant strains to produce hybrid plants; wherein said haplotypes are selected so that the first plant strain is heterozygous at both the S and Z loci, and said second plant strain is homozygous at one of the S and Z loci and heterozygous at the other of the S and Z loci; wherein said first and second plant strains are plants from the genera Lolium or Festuca; and wherein the first and second Z locus haplotypes are independently selected from a gene encoding a 26S proteasome subunit, or a no-pollen (NOP) polypeptide; and the said first and second S locus haplotypes are independently selected from a gene encoding a glutamate receptor or precursor thereof, or a seven-in-absentia homologue (SIAH).
 2. The method according to claim 1, wherein the plants are selected from the group consisting of Lolium perenne (perennial ryegrass), Lolium multiflorum (Italian ryegrass), Lolium boucheanum (hybrid ryegrass), Lolium arundinaceum (tall fescue) and Lolium pratense (meadow fescue).
 3. The method according to claim 1, wherein the Z locus haplotypes are selected from a gene encoded by one or more of: (a) the sequences shown in SEQ ID NOS: 58-59; (b) nucleotide sequences encoding the polypeptides shown SEQ ID NOS: 128-129; (c) complements of the full-length sequences recited in (a) and (b); (d) sequences antisense to the full-length sequences recited in (a) and (b); (e) functionally active fragments of the full-length sequences recited in (a), (b), (c) and (d) having at least 98% identity thereto and having a size of at least 100 nucleotides; and (f) functionally active variants of the sequences recited in (a), (b), (c), (d) and (e) having at least 98% identity thereto; and wherein the S locus haplotypes are selected from a gene encoded by one or more of: (a) the sequences shown in SEQ ID NOS:39-40; (b) nucleotide sequences encoding the polypeptides shown SEQ ID NOS:109-110; (c) complements of the full-length sequences recited in (a) and (b); (d) sequences antisense to the full-length sequences recited in (a) and (b); (e) functionally active fragments of the sequences recited in (a), (b), (c) and (d) having at least 98% identity thereto and having a size of at least 100 nucleotides; and (f) functionally active variants of the sequences recited in (a), (b), (c), (d) and (e) having at least 98% identity thereto. 